We recently developed a new approach to characterizing activity of receptor tyrosine kinases (RTK) on tumor cells by visualizing signaling associated protein complexes using proximity ligation assay technology. This was motivated by the (i) the poor performance of many RTK expression assays, such as IHC or FISH, that measure protein or gene expression rather than signaling activity, (ii) the need to better characterize active signaling events in tumor cells driven by tumor stromal mechanisms, (iii) the realization that some patients still benefit from anti-RTK directed therapy despite their tumors lacking genomic alterations, and (iv) the need to develop assays that can better identify mechanisms of acquired resistance to kinase inhibitor therapy and thus guide clinicians about subsequent treatment. Activation of RTK through diverse mechanisms, including gene mutation, gene amplification, or overproduction of activating growth factor ligands, leads to assembly of protein complexes on RTK that facilitate downstream signal transduction. Exploiting this fact, we have developed proximity ligation assays (PLA) to annotate protein complexes reflecting an activated EGFR. PLA can be developed starting with mass spectrometry based protein-protein interaction datasets to characterize in situ EGFR complexes reflecting active signaling in cancer tissues. We have developed one such assay reflecting EGFR in complex with its adaptor protein GRB2. We show these assays reflect EGFR kinase activity both in vitro using cancer cell lines and in vivo using mouse xenograft models of lung cancer. Importantly, these assays perform well in formalin fixed paraffin embedded (FFPE) tissues enabling wide spread utility in typical hospital tumor samples. We demonstrate feasibility and utility in characterizing EGFR:GRB2 complexes in nearly 300 patient derived xenograft models of cancer. Further, our data indicate good concordance of high PLA signals across three independent cohorts of lung cancer patients numbering 350 unique cases. Our human tumor data shows high PLA signal in tumors with activating EGFR mutation as expected but we can further identify tumors with high PLA signal that lack EGFR mutation or with KRAS mutation, thus representing tumor stromal activation of EGFR. We show these assays can detect EGFR driven resistance in lung cancers driven by EML4-ALK translocations and along with other published studies indicate a role of EGFR PLA assays to detect acquired resistance to kinase inhibitors driven by EGFR signaling. Our vision is that PLA technology can be used to define signaling associated protein complexes for many RTK beyond EGFR, including MET, FGFR, and AXL, which are primary targets of emerging therapeutics and also involved in resistance. This offers the future potential to have multiple PLA assays each reflecting signaling associated protein complexes for RTK matched to therapeutics. During the UH2 aim, we will conduct analytic validation studies of our existing EGFR PLA in a CLIA-compliant environment. During the UH3 aim, we will conduct clinical validation studies by investigating the association of PLA with response to EGFR-directed therapies.